During our characterization of the DNA-PKcs defect in SCID foals, we observed extensive splice variation within the region of the transcript encoding the phosphatidyl inositol 3 kinase (PI3K) domain. Completely analogous splice variation is also observed in human DNA-PKcs. One source of variation is the differential insertion of a single exon just proximal to the region of the transcript encoding the conserved protein kinase motifs. Additional variation is accomplished by intron retention. A significant percentage of DNA-PKcs transcripts retain a short intron within the PI3K domain resulting in the insertion of a stop codon prior to the purported catalytic site in the kinase domain. Transcripts retaining the intron would encode a truncated form of DNA-PKcs with a predicted mass of ~441kD (as opposed to ~469kD) which would theoretically lack protein kinase activity. This transcript is expressed in all species examined to date and is strongly conserved. We have preliminary evidence that this transcript is translated. Furthermore, analyses of DNA-PK activity in cells, which express high levels of this transcript, encodes a regulatory from of the kinase. Finally, expression of these DNA-PKcs splice variants is markedly higher in resting cells than in cycling cells suggesting a link between DNA-PKcs and cell cycle progression. The hypothesis of the proposed research is that since each DNA-PKcs transcript encodes a structurally distinct protein, each from will have distinct functions. The overall goal of the proposed research is to determine the function of each DNA-PKcs using both biochemical and genetic approaches.